August

Abstract

This time the mutation is successful, but we still don't finish building our vector.

Week 5

8.26.2012-8.31.2012

8.26.2012

Because the PCR production is OK, we know that we have built the right vector. So we picked up some colonies from the petri dish, cultured bacterium in shake-flask.

This process usually takes one night.

8.27.2012

We picked up the plasmid from the bacteria this morning, then digested it with two kinds of enzymes xba I and Pst I. Only in this way we can connect the terminator with the vector.

Despite the fact that we have already digested the vector to leave the restriction enzyme cutting site, we need to test the vector again. So we digested the vector with AflⅡ. If the vector is right, we should be able to see a stripe appear around 4kbp.
We have already digested the GFP, RFP and plasmid the day before yesterday. Today we need to do an electrophoresis to recover the gel, and get the purified GFP, RFP and plasmid.

Then we used T4 DNA ligase to connect GFP and the plasmid together.

During the 2 fragments ligation, we did an electrophoresis to see whether the vector ligased with 3 fragments successfully. We got disappointed that this vector is false positive which means we didn’t link GFP RFP and plasmid well.

We need to analysis why the experiment failed again. We think that the essence of our experiment is to digest the plasmid well. Because we used two different kinds of enzymes and a buffer to digest the plasmid, we can’t promise the efficiency of both enzymes are high enough. So we probably should spend more time on digestion next time.

After the discussion, we did the ligament（connection）again. We ligase GFP, RFP and the plasmid together with the system we used before. Two sets of ligaments were conducted: 1. connecting GFP and the plasmid at first, then connecting the product with RFP (mentioned as 2-fragments scheme later); 2. Connecting GFP, RFP, and the plasmid at the same time (mentioned as 3-fragments scheme).

8.28.2012

We’ve already did the 2-fragments and 3-fragments schemes yesterday. We need to transfer the artificial plasmid into the body of bacteria and let the E.coli grow.

8.29.2012

This morning, we took the petri dish out to see the colonies. There are many colonies growing on all the dishes which is not good news to us because it means this colonies doesn’t contain the vector we wanted. The plasmid may not contain GFP and RFP at all.
We don’t know why the experiment failed again. So we decided to check whether the problem came from those two different kinds of enzymes we used.

This time, we also did the 2-fragments and 3-fragments scheme connections at the same time. In order to ensure the efficiency of the restriction enzymes and T4 DNA ligase, we used only one kind of enzyme to digest and make sure the reaction time is long enough. We also did a control experiment to see whether the connection is successful. If the negative control grows well, the digestion is not successful.

8.30.2012

Today’s work is transformation. The process itself is not very difficult, but there are a lot of details we have to pay attention to. We did yesterday’s connection again and repeated the transformation.

All of us dreamed that the petri dish will give us hope.

8.31.2012

Biology laboratory is a place where strange things always happen.

We did two parallel experiments with the same condition and reaction system, but we get totally different results.

We are confused by the results and can’t find an acceptable explanation. It is doubtful what should be done next. So we only did a PCR experiment to check the results.